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UNLABELLED - Type I IFN signaling, which is initiated through activation of the alpha interferon receptor (IFNAR), regulates the expression of proteins that are crucial contributors to immune responses. Paramyxoviruses, including human metapneumovirus (HMPV), have evolved mechanisms to inhibit IFNAR signaling, but the specific contribution of IFNAR signaling to the control of HMPV replication, pathogenesis, and adaptive immunity is unknown. We used IFNAR-deficient (IFNAR(-/-)) mice to assess the effect of IFNAR signaling on HMPV replication and the CD8(+) T cell response. HMPV-infected IFNAR(-/-) mice had a higher peak of early viral replication but cleared the virus with kinetics similar to those of wild-type (WT) mice. However, IFNAR(-/-) mice infected with HMPV displayed less airway dysfunction and lung inflammation. CD8(+) T cells of IFNAR(-/-) mice after HMPV infection expressed levels of the inhibitory receptor programmed death 1 (PD-1) similar to those of WT mice. However, despite lower expression of inhibitory programmed death ligand 1 (PD-L1), HMPV-specific CD8(+) T cells of IFNAR(-/-) mice were more functionally impaired than those of WT mice and upregulated the inhibitory receptor Tim-3. Analysis of the antigen-presenting cell subsets in the lungs revealed that the expansion of PD-L1(low) dendritic cells (DCs), but not PD-L1(high) alveolar macrophages, was dependent on IFNAR signaling. Collectively, our results indicate a role for IFNAR signaling in the early control of HMPV replication, disease progression, and the development of an optimal adaptive immune response. Moreover, our findings suggest an IFNAR-independent mechanism of lung CD8(+) T cell impairment.
IMPORTANCE - Human metapneumovirus (HMPV) is a leading cause of acute respiratory illness. CD8(+) T cells are critical for clearing viral infection, yet recent evidence shows that HMPV and other respiratory viruses induce CD8(+) T cell impairment via PD-1-PD-L1 signaling. We sought to understand the role of type I interferon (IFN) in the innate and adaptive immune responses to HMPV by using a mouse model lacking IFN signaling. Although HMPV titers were higher in the absence of type I IFN, virus was nonetheless cleared and mice were less ill, indicating that type I IFN is not required to resolve HMPV infection but contributes to pathogenesis. Further, despite lower levels of the inhibitory ligand PD-L1 in mice lacking type I IFN, CD8(+) T cells were more impaired in these mice than in WT mice. Our data suggest that specific antigen-presenting cell subsets and the inhibitory receptor Tim-3 may contribute to CD8(+) T cell impairment.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Human metapneumovirus (HMPV) is a major cause of respiratory disease. The role of NK cells in protection against HMPV is unclear. We show that while HMPV-infected C57BL/6 mice had higher numbers of functional lung NK cells than mock-treated mice, comparing NK cell-depleted and control mice did not reveal differences in lung viral titers, histopathology, cytokine levels, or T cell numbers or function. These data indicate that NK cells are not required for host control of HMPV.
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
BACKGROUND - The inpatient and outpatient burden of human metapneumovirus (HMPV) infection among young children has not been well established.
METHODS - We conducted prospective, population-based surveillance for acute respiratory illness or fever among inpatient and outpatient children less than 5 years of age in three U.S. counties from 2003 through 2009. Clinical and demographic data were obtained from parents and medical records, HMPV was detected by means of a reverse-transcriptase polymerase-chain-reaction assay, and population-based rates of hospitalization and estimated rates of outpatient visits associated with HMPV infection were determined.
RESULTS - HMPV was detected in 200 of 3490 hospitalized children (6%), 222 of 3257 children in outpatient clinics (7%), 224 of 3001 children in the emergency department (7%), and 10 of 770 asymptomatic controls (1%). Overall annual rates of hospitalization associated with HMPV infection were 1 per 1000 children less than 5 years of age, 3 per 1000 infants less than 6 months of age, and 2 per 1000 children 6 to 11 months of age. Children hospitalized with HMPV infection, as compared with those hospitalized without HMPV infection, were older and more likely to receive a diagnosis of pneumonia or asthma, to require supplemental oxygen, and to have a longer stay in the intensive care unit. The estimated annual burden of outpatient visits associated with HMPV infection was 55 clinic visits and 13 emergency department visits per 1000 children. The majority of HMPV-positive inpatient and outpatient children had no underlying medical conditions, although premature birth and asthma were more frequent among hospitalized children with HMPV infection than among those without HMPV infection.
CONCLUSIONS - HMPV infection is associated with a substantial burden of hospitalizations and outpatient visits among children throughout the first 5 years of life, especially during the first year. Most children with HMPV infection were previously healthy. (Funded by the Centers for Disease Control and Prevention and the National Institutes of Health.).
Paramyxoviruses use a specialized fusion protein to merge the viral envelope with cell membranes and initiate infection. Most paramyxoviruses require the interaction of two viral proteins to enter cells; an attachment protein binds cell surface receptors, leading to the activation of a fusion (F) protein that fuses the viral envelope and host cell plasma membrane. In contrast, human metapneumovirus (HMPV) expressing only the F protein is replication competent, suggesting a primary role for HMPV F in attachment and fusion. We previously identified an invariant arginine-glycine-aspartate (RGD) motif in the HMPV F protein and showed that the RGD-binding integrin αVβ1-promoted HMPV infection. Here we show that both HMPV F-mediated binding and virus entry depend upon multiple RGD-binding integrins and that HMPV F can mediate binding and fusion in the absence of the viral attachment (G) protein. The invariant F-RGD motif is critical for infection, as an F-RAE virus was profoundly impaired. Further, F-integrin binding is required for productive viral RNA transcription, indicating that RGD-binding integrins serve as receptors for the HMPV fusion protein. Thus, HMPV F is triggered to induce virus-cell fusion by interactions with cellular receptors in a manner that is independent of the viral G protein. These results suggest a stepwise mechanism of HMPV entry mediated by the F protein through its interactions with cellular receptors, including RGD-binding integrins.
Viruses are leading causes of severe acute lower respiratory infections (LRIs). These infections evoke incomplete immunity, as individuals can be repeatedly reinfected throughout life. We report that acute viral LRI causes rapid pulmonary CD8+ cytotoxic T lymphocyte (TCD8) functional impairment via programmed death-1/programmed death ligand-1 (PD-1/PD-L1) signaling, a pathway previously associated with prolonged antigenic stimulation during chronic infections and cancer. PD-1-mediated TCD8 impairment occurred acutely in mice following infection with human metapneumovirus or influenza virus. Viral antigen was sufficient for PD-1 upregulation, but induction of PD-L1 was required for impairment. During secondary viral infection or epitope-only challenge, memory TCD8 rapidly reexpressed PD-1 and exhibited severe functional impairment. Inhibition of PD-1 signaling using monoclonal antibody blockade prevented TCD8 impairment, reduced viral titers during primary infection, and enhanced protection of immunized mice against challenge infection. Additionally, PD-1 and PD-L1 were upregulated in the lungs of patients with 2009 H1N1 influenza virus, respiratory syncytial virus, or parainfluenza virus infection. These results indicate that PD-1 mediates TCD8 functional impairment during acute viral infection and may contribute to recurrent viral LRIs. Therefore, the PD-1/PD-L1 pathway may represent a therapeutic target in the treatment of respiratory viruses.
BACKGROUND - Human metapneumovirus (HMPV) is a paramyxovirus with multiple genetic lineages that is a leading cause of acute respiratory disease. Several RT-PCR assays have been described based on limited available sequence data.
OBJECTIVES - To develop a broadly reactive real-time RT-PCR assay for HMPV that allows for a rapid, sensitive, and specific detection in a clinical or research setting.
STUDY DESIGN - Three published assays for HMPV were modified based on analysis of multiple HMPV sequences obtained from GenBank. Original and modified assays were tested against prototype HMPV strains from each genetic sublineage, multiple isolates of HMPV from different years, a collection of clinical specimens, and commercial validation panels.
RESULTS - A number of potential sequence mismatches with diverse HMPV strains were identified. Modifications were made to oligonucleotides to improve annealing efficiency. Primers and probes based on newer sequence data offered enhanced detection of all subgroups, especially for low titer specimens. The new primers and probe detected multiple clinical isolates of HMPV collected over a twenty-year period. The modified assay improved detection of HMPV in a panel of clinical specimens, and correctly identified HMPV samples in two commercial validation sets.
CONCLUSIONS - We report a modified real-time RT-PCR assay for HMPV that detects all genetic lineages with high sensitivity.
Copyright © 2012 Elsevier B.V. All rights reserved.
BACKGROUND - We performed a prospective study to determine the disease burden of respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) in older adults in comparison with influenza virus.
METHODS - During 3 consecutive winters, we enrolled Davidson County (Nashville, TN) residents aged ≥ 50 years admitted to 1 of 4 hospitals with acute respiratory illness (ARI). Nasal/throat swabs were tested for influenza, RSV, and HMPV with reverse-transcriptase polymerase chain reaction. Hospitalization rates were calculated.
RESULTS - Of 1042 eligible patients, 508 consented to testing. Respiratory syncytial virus was detected in 31 participants (6.1%); HMPV was detected in 23 (4.5%) patients; and influenza was detected in 33 (6.5%) patients. Of those subjects aged ≥ 65 years, 78% received influenza vaccination. Compared with patients with confirmed influenza, patients with RSV were older and more immunocompromised; patients with HMPV were older, had more cardiovascular disease, were more likely to have received the influenza vaccination, and were less likely to report fever than those with influenza. Over 3 years, average annual rates of hospitalization were 15.01, 9.82, and 11.81 per 10,000 county residents due to RSV, HMPV, and influenza, respectively.
CONCLUSIONS - In adults aged ≥ 50 years, hospitalization rates for RSV and HMPV were similar to those associated with influenza.
Human metapneumovirus and respiratory syncytial virus cause lower respiratory tract infections. The virus fusion (F) glycoprotein promotes membrane fusion by refolding from a metastable pre-fusion to a stable post-fusion conformation. F is also a major target of the neutralizing antibody response. Here we show that a potently neutralizing anti-human metapneumovirus antibody (DS7) binds a structurally invariant domain of F, revealing a new epitope that could be targeted in vaccine development.
Human respiratory syncytial virus (RSV) and human metapneumovirus (MPV) are two of the most common causes of serious viral lower respiratory tract illness in humans. CD8+ T cells have been shown to be important in animal models and human clinical studies for the clearance of viral infection, and they may contribute in part to protection against severe disease during reinfections. Precise enumeration and accurate phenotyping of RSV- or MPV-specific CD8+ T cells in humans is currently limited by the relatively small number of T cell epitopes that have been mapped with accompanying identification of MHC restriction patterns. We sought to expand the number of potential RSV and MPV epitopes for use in clinical and translational studies by identifying an expanded set of MHC-binding peptides based on RSV and MPV wild-type virus strain protein sequences. We interrogated the full protein sequences of all 9 or 11 proteins of MPV or RSV respectively using four established epitope prediction algorithms for human HLA A*0101, A*0201, or B*0702 binding and attempted to synthesize the top-scoring 150-152 peptides for each of the two viruses. Synthesis resulted in 442 synthesized and soluble peptides of the 452 predicted epitopes for MPV or RSV. We then determined the binding of the synthetic peptides to recombinant human HLA A*0101, A*0201 or B*0702 molecules with the predicted restriction using a commercially available plate-based assay, iTopia. A total of 230 of the 442 peptides tested exhibited binding to the appropriate MHC molecule. The binding results suggested that existing algorithms for prediction of MHC A*0201 binding are particularly robust. The binding results also provided a large benchmarking data collection for comparison of new prediction algorithms.
Copyright © 2011 Elsevier B.V. All rights reserved.